C 0V A L EN T S T R U C T U R E 0F 7 G
- I M M U N O G L O B U L IN.
Light, A. (1967b), Methods Enzymol. 11,426. Natvig, J. B., and Kunkel, H. G. (1968), Ser. Haematol. 1,66. Prahl, J. W. (1967), Biochem. J . 105,1019. Rutishauser, U., Cunningham, B. A,, Bennett, C., Konigsberg, W. H., and Edelman, G. M. (1968), Proc. Nut. Acad. Sci. U.S. 61,1414. Schroeder, W, A. (1967), Methods Enzymol. 11,361. Schroeder, W. A. (1969), Arch. Biochem. Biophys. 130, 551. Singer, S . J., and Doolittle, R. F. (1966), Science 153,13.
I X
Smyth, D. G. (1967), Methods Enzymol. I I , 421. Thorpe, N. O., and Deutsch, H. F. (1966), Immunochemistry 3,329. Wang, A., and Fudenberg, H. H. (1969), J . Mol. Biol. 44,493. Waxdal, M. J., Konigsberg, W. H., and Edelman, G . M. (1968b), Biochemistry 7,1967. Waxdal, M. J., Konigsberg, W. H., Henley, W. L., and Edelman,G. M. (1968a), Biochemistry 7,1959. World Health Organization (1964), Bull. W . H . 0.30,447.
The Covalent Structure of a Human yG-Immunoglobulin. IX. Assignment of Asparaginyl and Glutaminyl Residues* Carl Bennett,? William H. Konigsberg, and Gerald M. Edelman$
The locations of the asparaginyl and glutaminyl residues in the amino acid sequence of the human yG1-immunoglobulin Eu have been determined by subjecting the
ABSTRACT:
S
tudies on the determination of the amino acid sequence of the rG1-immunoglobulin Eu have been reported in the preceding papers of this series (Cunningham et al., 1968; Gottlieb et al., 1970; Cunningham et al., 1970; Rutishauser et al., 1970). The methods chosen for analysis of the amino acid sequences and compositions of peptides required the use of acid hydrolysis, a procedure which destroys amides. As a result, the position of asparaginyl and glutaminyl residues could not be determined directly. The purpose of this paper is to present the evidence used to establish the positions of these residues. Peptides which had been isolated previously were submitted to paper electrophoresis for determination of their mobilities or were degraded under conditions which maintained the integrity of asparaginyl and glutaminyl residues. Certain peptides contained a large number of Asx1 and Glu residues and therefore had to be subjected to special treatment. Materials and Methods The isolation and purification of the peptides used have been described in previous papers of this series (Cunningham et al., 1968; Gottlieb et al., 1970; Cunningham et al., 1970; Rutishauser et al., 1970).
* From the Department of Biochemistry, Yale University, New Haven, Connecticut, and The Rockefeller University, New York, New York. Receiued December 8, 1969. This work was supported in part by grants from the National Science Foundation (GB 6655 and GB 8371) and the U. S. Public Health Service (GM 12607, AM 04256, and AI 09273). t Postdoctoral Fellow of the U. S. PublicHealth Service. Reprint requests may be addressed to G. M. Edelman, The Rockefeller University, New York, N. Y.10021. Abbreviations used that are not listed in Biochemistry 5, 1445 (1966), are: Asx, aspartic acid or asparagine; Glx, glutamic acid or glutamine.
purified peptides to high-voltage paper electrophoresis or to complete enzymatic hydrolysis followed by amino acid analysis.
Leucine aminopeptidase, a-chymotrypsin, subtilisin, DFPtreated carboxypeptidase A, and carboxypeptidase B were obtained from Worthington Biochemical Corp., Freehold, N. J. Pronase was obtained from Calbiochem, Los Angeles, Calif. Aminopeptidase M was purchased from Henley and Company, New York, N. Y.Chymotrypsin C was a gift from J. E. Folk and streptococcal proteinase was a gift from S. Moore and W. H. Stein. All chemicals used were reagent grade except for pyridine which was refluxed with ninhydrin and distilled before use. Enzymatic Digestions:
1. LEUCINE AMINOPEPTIDASE. A solution of the enzyme (0.5 mg/ml) in 5 mM sodium barbital buffer425 mM in MgCl2, pH 8.5, was dialyzed against distilled water for 1 hr. A 100-pl aliquot of the enzyme solution was added to approximately 30 nmoles of the peptide in 100 pl of 50 mM sodium barbital buffer-2.5 mM MgC12, pH 8.5. The mixture was incubated at 37" and digestion was terminated after 3 hr by the addition of 700 p1 of 0.3 M lithium citrate buffer, pH 2.2. 2. PRONASE AND AMINOPEPTIDASE M. A solution of pronase (0.5 mg/ml, 100 pl) was added to 30 nmoles of the peptide in 100 p1 of 50 mM sodium barbital buffer, pH 7.7. The mixture was incubated for 16 hr at 37". A solution of aminopeptidase M (0.5 mg/ml, 10 pl) in the same barbital buffer was then added and the mixture was incubated for 4 hr at 37". The digestion was terminated by addition of 0.3 M lithium citrate buffer, pH 2.2. 3. CARBOXYPEPTIDASES A AND B. A stock solution of a mixture of carboxypeptidase A and carboxypeptidase B was prepared by dissolving 50 p1 of each enzyme suspension in 500 pl of 1 M lithium chloride and adding 500 p1 of 1 M NaHCOa. The solution was dialyzed against 0.01 M NaHC08 for 2 hr. This
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Gln-Pro- Asp-AspPhe LT7C3C
108 110 115 120 125 Gly-Thr-Val-Ala-Ala-Pro-Set-VaI-Ph~Ile-Phe-Pro-Pro-Ser-AspGlu-GIn-Leu-Lys LTlO I
I
137 140 145 Asn-Asn.Phe-Tyr-Pro-ArgGlu-Ala-Lys-VaI- Gln-Trp LC14
150 155 160 165 VaI-Asp-Asn-Ala-Leu-Gln-Ser-Gly-AsnSer-GIn-Glu-Ser-Val-Thr-Glu-GIn-AspSerCys LT14 I
1
LT14CC3 p
-
LT14CC 2
LT14 CC I
LTi4CC4
I+-----!
t
t
A
I
E
c-------l
w
A , ,
E
F
1 : Amino acid sequence of peptides from the Eu light chain. Numbers above the sequence indicate the positions in the amino acid sequence from the NH2 terminus of the chain (Cunningham et al., 1968; Gottlieb et al., 1970). Peptides LT14CC1 through LT14CC4 (Figure 1D) were obtained from a chymotrypsin C digest of LT14. Peptides A through F (Figure 1D)were obtained from a digest of LT14CCI with streptococcal proteinase. FIGURE
stock solution was stable for 3 weeks at -4'. The stock solution (50 pl) was added to about 30 nmoles of the peptide in 200 pl of 0.05 M sodium barbital buffer, pH 8.5. For digestion of large peptides, sodium dodecyl sulfate was added to a final concentration of 0.08 %. The digestion was carried out at 35 for various lengths of time, and was terminated by the addition of 600 pl of 0.3 M lithium citrate buffer, pH 2.2. The solutions were centrifuged and the amino acid compositions of the supernatants were determined directly in the amino acid analyzer. 4. CHYMOTRYPSIN. Chymotrypsin (1 %, w/w) was added to a solution of the peptide in 1 ml of 0.1 M NH4HC03.Digestion was carried out at 37 O for 3 hr and was terminated by lyophilization. 5. CHYMOTRYPSIN c. A solution of chymotrypsin C (1 mg/ml in 0.1 M N H I H C O ~pH , 8.0) corresponding to 1 % (w/w) of the peptide and 1 mg of 1,lO-phenanthrolinehydrochloride (Toda and Folk, 1969) were added to the peptide in 1 ml of 0.1 M NHrHC03,pH 8.0. After 4 hr at 25Othe digestion was terminated by lyophilization. 6. DIGESTION WITH STREPTOCOCCAL PROTEINASE AND SUBTILISIN. Digestion was performed according to procedures previously described (Gerwin et al., 1966; Weber and Konigsberg, 1967). Paper electrophoresis was performed in tanks under varsol (Savant, Hicksville, N. Y.)at pH 6.5 (pyridine-acetic acidwater, 25:1:375, v/v), and at pH 4.7 (pyridine-acetic acidwater, 1 :1 :38, v/v) and pH 1.9 (formic acid-acetic acid-water, 22:87:1871, v/v) (Schwartz and Edelman, 1963). A known mixture of amino acids (10 nmoles) was used as a standard.
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Peptides were located by spraying with 0.025 % ninhydrin in 95 ethanol. The mobility of each peptide relative to that of aspartic acid was measured, and the net charge on the peptide was calculated from this mobility and from the known molecular weight (Offord, 1966). Electrophoresis of peptides containing histidine was carried out at both pH 6.5 and pH 4.7 because the imidazole ring is only partially charged at 6.5 but becomes fully charged at pH 4.7. For preparative purposes, acidic and neutral peptides were eluted from the paper with 50% pyridine and basic peptides were eluted with 50% acetic acid. Zon-Exchange Chromatography. Separation of the peptides produced by digestion with chymotrypsin C or subtilisin was carried out on columns of AG5OX4 (Cunningham et al., 1968). Other buffer gradients have also been described (gradients I and 11, Weber and Konigsberg, 1967). Amino .4cid Analysis. Amino acid analyses were carried out as previously described (Edelman et a/., 1968) except that asparagine and glutamine were determined using the lithium citrate system (Benson et al., 1967). The dansyl-Edman (Cunningham et al., 1968) and the Edman (Jeppson and Sjoquist, 1967) techniques have previously been described. Results and Discussion Two principal methods were used in the present studies: high-voltage paper electrophoresis and amino acid analysis after prolonged enzymatic digestion. An estimate of the net charge of a peptide can be obtained from its electrophoretic mobility and molecular weight (Offord, 1966). At pH 6.5, aspartyl and glutamyl residues are negatively charged while asparaginyl and glutaminyl residues are uncharged; thus the net charge may be used as a measure of the number of acidic side chains in the peptide. Alternatively, the peptide may be subjected to prolonged enzymatic hydrolysis with pronase and aminopeptidase M. This procedure preserves asparaginyl and glutaminyl residues, which may then be measured by amino acid analysis (Benson et ai., 1967). One or both of these methods were usually sufficient for assignment of the positions of asparaginyl and glutaminyl residues. For some peptides, however, enzymatic digestion with leucine aminopeptidase or carboxypeptidase or degradation by the Edman technique were necessary, The results of all of the studies are summarized in Tables I, 11, and 111. In most cases, the assignments are self-evident and require no further discussion. Peptides which presented particular difficulties will be discussed separately for light and heavy chains. Isolation and placement of the light-chain peptides have been described by Cunningham et ai. (1968) and Gottlieb et ai. (1970). Similar descriptions of the heavy-chain peptides have been given by Cunningham et al. (1970) and Rutishauser et al. (1970). Light Chain. Data used for the assignment of asparaginyl and glutaminyl residues in the light chain are summarized in Table I. Four regions of the light-chain sequence required special attention ; the sequences of these regions are presented in Figure 1. An acidic peptide (LT7C3C) with the composition Glx, 1.1; Pro, 1.1; Asx, 1.9; Phe, 0.8 (Figure IA, residues 79-83) was isolated by high-voltage paper electrophoresis at pH 6.5 after extensive chymotryptic digestion of peptide LT7C3. The
COVALENT STRUCTURE OF 'YG-IMMUNOGLOBULIN. IX
TABLE I :
Assignment of Amides in the Light Chain.
Residue Number
1 3 6 17 27 30 37 38 55
70 79' 81' 821 89 90 92 94 100 105 1221 1231 124' 1371 1381 1431 147' 1511 1521 155' 1581 1601 161' 1651 1661 1671 170 185 187 195 199 210 21 3
Result Asp. Gln Gln ASP Gln Asn Gln Gln Glu Glu Gln ASP ASP Gln Gln Asne ASP Gln Glu ASP Glu GM Asna Asn Glu Gln Aspc Asn Gln Asn Gln Glu Glu Glni ASP ASP Asp Glu Glu Gln Asn Glu
Residues
Net Charge at pH 6.5.
Peptides Used
Spanned
Found
LT1 LT1 LTl LT1 LT3C1 LT3Cl LC6 LC6 LT6 LT7C1 LT7C3C LT7C3Cd LT7C3Cd LCI 1 k LC1 lk LT7C5 LT7C5 LT8 LT9 LTlO LTlO LTlO LC14 LC14 LC14 LC14 LT14CC30 LTl4CC30 LT14CC4 LT14CC4 LTl4CC1C* LT14CClD LT14CC1F h LT14CClp LT14CC20 LTl5 LT16 LT16 LC19SP1' LC19SP2j LT19 LT20k
1-18 1-18 1-18 1-18 25-32 25-32 36-48 36-48 51-61 62-71 79-83 80-83 80-83 87-91 87-91 92-96 92-96 97-103 104-107 108-1 26 108-126 108-126 137-148 137-148 137-148 137-148 150-1 54 150-1 54 155-1 58 155-158 159-1 60 161-162 165-1 66 159-166 167-1 69 170-183 184-1 88 184-188 193-1 97 198-201 208-21 1 2 12-214
Calculated
Amino Acid Analysisb Asp
Asn
1.5
0
f 3 (0.59) 0
-2 (0.72) -2 (0.82)
0
0.1
1.0
-2 (0.69) -2 (0.80)
0
0.8
0
0.7
0.9 0.8
0
0 -1 (0.70) -1 (0.58)
1.6
0
0.1
0
0.2
1.5
0
-1 (0.38)
Gln
0.1
+3 (0.58)
0
$1 (0.36) 0 -1 (0.15)
Glu
0.1 0.8
+1 (0.32) 0 -1 (0.17)
-1 (0.41)
1.4
0.9 0.8
0.1
1.5
0.8
0.8
0.9
0.8
0.8
0.7
0.8
0.7
0
0 -1 (0.71) -1 (0.63) 0
0.9 0.9 0.9
0.8 0.9 0.7
+1 (0.44) -1 (0.51)
+1 (0.41) -1 (0.52)
0.9
The numbers in parentheses are the measured and calculated mobilities of each peptide. b Numbers in each column indicate the moles of amino acid obtained per mole of peptide after enzymatic digestion. Identified as PTH-Asp. d Peptide was obtained after one step of the Edman degradation of LT7C3C. Identified as PTH-Asn. f Determined by treatment of LTlO with carboxypeptidases A and B. Peptides were obtained from a chymotrypsin C digest of LT14 (residues 150-169). h Peptides were isolated from a streptococcal proteinase digest of LTl4CCl (residues 159-166). i This assignment was based on the release of glutamine when LT14CCl was digested with carboxypeptidase A. Obtained from a streptococcal proteinase digest of LC19. k The cysteinyl residue is blocked with a carboxamidomethyl group. 1 The assignment of this residue is discussed in the text. 0
6
net charge of peptide LT7C3C was -2 (Table I). One step of the Edman degradation yielded FTH-Gln and the resulting tetrapeptide (residues 80-83, Table I) still had a net charge of
-2, indicating that both residues 81 and 82 were aspartic acid. Digestion of peptide LTlO (residues 108 to 126, Figure 1B)
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I 12 PCA~VJI.Gln.Le~VoI.GIn-SerGly-AlaGlu.Vol.Lys
HTI
I
I
48
39
Gln-Alof ro.Gly-Gln.Gly.Leu-Glu.Trp.Mel HT60 I -I
81
73
AspGlu~Ser-Thr-As~Thr-Alo~Tyr.Mel
HTBo-LAP
I
I
CI
I
96 109 Cys~Alo~Gly~Gly-TyrGly-Ile-Tyr-Ser-Pro~Glu~Glu~Tyr.Asn
HT9Ca
I
I
A5700*4* 0.2
50
IO0
150
200
ml Effluent FIGURE 2: Chromatography on AG50X4. (A) Ion-exchange chromatography of peptides obtained after digestion of peptide LT14 with chymotrypsin C. Gradient I was used (see Materials and Methods). (B) Chromatography of material from fraction I (Figure 2A) using gradient 11. (C) Chromatography of peptides obtained after digestion of fraction 4 (Figure of 2B) with streptococcal proteinase. Gradient I1 was used.
with pronase and aminopeptidase M released one residue each of aspartic acid, glutamic acid, and glutamine. The electrophoretic mobility of LTlO (Table I) is consistent with this composition. Treatment of the peptide with carboxypeptidases A and B yielded lysine, leucine, and glutamine. From these data, we conclude that residue 124 (Figure 1B) is glutamine and that residues 122 and 123 are aspartic acid and glutamic acid, respectively. Treatment of the peptide LC14 (residues 137 to 148, Figure IC) with pronase and aminopeptidase M indicated that it contained two residues of asparagine and one residue each of glutamic acid and glutamine (Table I). Residues 137 and 138 therefore are asparagine. Residue 137 was also identified as PTH-asparagine after one step of the Edman degradation. Digestion of LC14 with carboxypeptidase A released 0.6 residue of tryptophan and 0.8 residue of glutamine, indicating that position 147 is Gln. Residue 143 must therefore be glutamic acid. The assignment of an amide or a free acid to each of the nine positions in peptide LT14 (residues 150 to 169, Figure 1D) was made on the basis of analyses of smaller peptides derived from this tryptic peptide. A mixture of peptides from a chymotrypsin C digest of peptide LT14 was partially resolved by chromatography on AG50X4 (Figure 2A). Fraction 2 contained a single peptide, LT14CC2 (Asx, 1.0; Ser, 1.1; Lys, 0.9; Figure 1D). The sequence of this peptide was determined as Asx-Ser-Lys by the subtractive Edman procedure. Peptide LT14CC2 was neutral at pH 6.5 and released one residue of aspartic acid after treatment with aminopeptidase M (Table I), indicating that residue 167 is aspartic acid. The material from fraction I (Figure 2A) was further purified by ionexchange chromatography on AG50X4 (Figure 2B). Fraction
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148 158 Asp.Tyc.PhePro-Glu-Pra-Val-Thr.Vol-Sef-Trp HT12CI I f 256 '265 274 Thr~Pra-Glu-Val-Th~Cys~Val-Vol~Vol~Asp~~l~Ser~i~Glu-AspPra~ln~Vol-Lys HTI8
I----
SP I +-------+
I
I
SP3 +-----+
SP2
HTIEc
276 285 Asn-Trp.Tyr-VoI.Asp-Gly .VoI-Gln-Voi-His H5C2
4
I
H5C20
c----i
37 I 380 392 G ly-Phe-Tyr-Pro-Ser-A~p-Ile-Alo.Vol-Glu-Trp~Glu-Ser-Asn-~~Gly-Glu-~a~Glu-Asn-Tyr~Lys
HT33
I
I
i
cc I
-
I
t
CI
cc 2
t---------(
i
cc5 H
cc3
cc 4
41.7 420 430 439 Trp~Gln-Gln-Gly-Asn-Val-Phe-Ser-Cys-Ser-Vol-Mel-His-G'u-Ala-Leu-His-AsnHis-Tyr-Thrtln-Lys H i 37
H137 bCI c----i
HT37bC3 *
Amino acid sequence of peptides from the Eu heavy chain. Numbers above the sequence indicate the positions in the amino acid sequence from the NH2 terminus of the chain (Cunningham et al., 1970; Rutishauser et al., 1970). Peptides SPl through SP4a (Figure 3F) were obtained from a digest of HT18 with streptococcal proteinase. Peptides HT33CC1 through HT33CC5 were obtained from a digest of HT33 with chymotrypsin C. Peptide HT33C1 was obtained from a digest of HT33 with chymotrypsin. FIGURE 3:
3 from this chromatogram yielded peptide LT14CC3 (Asx, 1.9; Ala, 0.9; Val, 1.0; Leu, 1.1; Figure 1D). High-voltage electrophoresis of this peptide at pH 6.5 indicated that it contained one acidic residue, and digestion with amino-
C O V A L E N T S T R U C T U R E OF Y G - I M M U N O G L O B U L I N .
~~
I X
~~
~
TABLE 11: Assignment of Amides in CNBr Fragments HI through Ha.
Residue Number
Result
3. 9 l@ 390 430 49 59 62 65 73. 74 770 82 89 90 106 107 1090 1480 1520 159 175 196 201 203 208 212 216 22 1 233 249
Gln Gln Glu Gln Gln Glu Asn Gln Gln ASP Glu Asn Glud Glu ASP Glud Glud Asn ASP Glu Asn Gln Gln Asn Asn Asn ASP Glu ASP Glu ASP
Residues
Net Charge at pH 6.5.
Peptides Used
Spanned
Found
HT1 HT1 HlClP2 HT6a HT6a HT6a HT6c HT6c HT7 HT8a-LAP HT8a-LAP HT8a-LAP HT8b H4P1 H4P1 HT9C3 HT9C3 HT9Ca HTl2Cl HTl2Cl HTl2C2 HT12C4 HT12C5 HT12C6 HT12C6 HT12C6 HTI 3a HT14 HT15 H-H-2f HT17
1-12 1-12 5-10 39-48 39-48 39-48 55-63
55-63 64-67 73-81 73-81 73-81 82-87 87-93 87-93 104-1 2 1 104-1 21 96-109 148-158 148-1 58 159-1 68 175-180 181-198 199-2 10 199-210 199-210 211-213 215-218 219-222 223-241 240-255
Amino Acid Analysisb
Calculated
-1 (0.37)
-1 (0.38)
+1 (0.26)
+ l (0.26)
+1 (0.45) -2 (0.52)
+ I (0.42) -2 (0.50)
-2 (0.37) -2 (0.34)
-2 (0.36) -2 (0.41)
0 0 f 3 (0.63)’
0 0 +3 (0.61)
Asp
Asn
Glu
Gln
0.7
2.2
0.9 0.9
0.8 1.9 0.6
C
0.9 0.8
0.9
0.7
0.8
0.9
0.9
c
0.9
0 0 0 Od
0
0 0 0 0 0
0.9 0.8
2.7
0.7
The numbers in parentheses are the measured and calculated mobilities of each peptide. b Numbers in each column indicate the moles of amino acid obtained per mole of peptide after enzymatic digestion. c This residue was not released by enzymatic digestion, presumably because it is adjacent to a prolyl residue. d Identified as PTH-Glu. 8 Electrophoresis was carried out at pH 4.7 because histidine was present. f Gall et al. (1968). 0 The assignment of this residue is described in the text. 0
peptidase M followed by amino acid analysis showed that aspartic acid and asparagine were present in equal quantity. Two steps of the Edman degradation yielded PTH-Val followed by PTH-Asp. These results indicate that residue 151 is aspartic acid and that residue 152 is asparagine. Fraction 5 (Figure 2B) yielded peptide LT14CC4 (Asx, 0.9; Ser, 1.2; Glx, 0.9; Gly, 1.1; Figure 1D). Digestion of this neutral peptide (Table I) with aminopeptidase M released 0.8 residue of glutamine and 0.7 residue of asparagine. Therefore residue 155 is glutamine and residue 158 is asparagine. Peptide LT14CC1 (residues 159-166, Figure 1D) was obtained from fraction 4 (Figure 2B). In order to obtain smaller peptides comprising this region, peptide LT14CC1 was digested with streptococcal proteinase. Separation of this digest by ion-exchange chromatography on AG50X4 (Figure 2C) yielded peptides A through F (Figure 1D). Peptides A, B, and C (Figure 1D) were obtained by high-voltage paper
electrophoresis of fraction 3 (Figure 2C) at pH 1.9. Peptides D and F were obtained from fractions 1 and 2, respectively. Fraction 4 contained peptide E. The amino acid sequences of peptides A through E were determined by the dansyl-Edman procedure. The net charges of peptides C and D (Table I) indicate that residue 160 is glutamine and residue 161 is glutamic acid. Peptide F yielded only glutamic acid after acid hydrolysis, but its net charge (Table I) indicated that it contained one residue each of glutamic acid and glutamine. Positive Edman degradation liberated PTH-glutamic acid, indicating that residue 165 is glutamic acid and residue 166 is glutamine. This result was confirmed by digestion of LT14CC1 with carboxypeptidase A. These data complete the assignment of amides in peptide LT14 (Figure 1D). Heauy Chain. The data used for the assignment of the positions of glutamic acid, glutamine, aspartic acid, and asparagine in the heavy chain are presented in Tables I1 and 111.
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Assignment of Amides in CNBr Fragments H Sthrough H7.
Residue Number 2580 2650 2690 2700 2720 2760 2800 2830 286 293 294 295 297 311 312 315 318 325 333 342 3450 3470 356 357 361 362 3760 3800 3820 3840 3850 3870 3890 3900 399 401 41 3 4180 4190 4210 4300 434. 4380
KONIGSBERG,
Result Glu ASP Glu ASP Gln Asn ASP Gln Asn Glug Gln Gln Asxh Gln Asn ASP Glu Asn Glu Gln Glu Gln Glu Glu Asn Gln ASP Glu Glu Asn ASP Glug Gluo Asn ASP ASP ASP Gln Gln Asn Glu Asn Gln
Net Charge at pH 6.5.
-
Peptides Used
Residues Spanned
Found
Calculated
HT18SPlc HT18SP2c HT18SP3c HT18SP3c HT18cJ H5C2a H5C2 H5C2 H5C3T1 HT2 1 HT2lAl HT2lAl HT21 H5C5a H5C5a H5C5b HT23 HT25 HT26 HT29 HT30Clt HT30Clj H04ak H04ak HT32SP1 HT32SPl' HT3 3CC1m HT3 3CC2n HT33CC3n HT33CC4n HT33Cl. HT33Cln HT33Cl. HT33CC5m HT34a HT34a HT35 HT37 HT37 HT37 HT37bC1 HT37 HT37bC3
256-260 262-265 268-272 268-272 271-274 276-278 276-285 276-285 286-288 293-301 293-296 293-296 293-301 307-313 307-313 3 14-3 19 318-320 323-326 327-334 341-344 345-349 346-349 350-3 58 350-358 361-365 361-365 371-378 378-381 379-382 379-384 382-392 382-392 382-392 390-392 393-404 393-404 410-414 417-439 417-439 417-439 429-432 417-439 437439
-1 (0.42)
-1 (0.40)
-1d (0,34)
-1 (0.37)
+1 (0.33)
+1 (0.42)
0
Od
Amino Acid Analysis* Asp
Asn
0.7 e
0.9
Glu
Gln
0.9
e
0.9
1.9
0.9 0.9 0.8
+ld
(0.17)
-1 (0.37) 0
$1 (0.29) -1 (0.33)
0.6
0.4
0.8 1.0
0.8
0
0.7 0 +1 (0.47) -1 (0.38) 0
0 +1 (0.45) -1 (0.36)
0.8
0
1.7 0
0
0.9
0.8
0.8 0.6 -3 (0.63)
-3 (0.64)
+1 (0.47) -2 (0.43)
+1 (0.48) -2 (0.44)
0
Od
0
0.8 1.7 1.7
0.8 0.8 2.0
1 .o
2.0
0.8 1. o
0 ~-
3.0
3.0 0.9 ~
The numbers in parentheses are the measured and calculated mobilities of each peptide. * Numbers in each column indicate the moles of amino acid obtained per mole of peptide after enzymatic digestion. c These peptides were derived from a streptococcal proteinase digest of HT18 (residues 256-274). d Electrophoresis was carried out at pH 4.7 because histidine was present. e This residue was not released by enzymatic digestion, presumably because it is adjacent to a prolyl residue. Obtained from a tryptic digest of Fc(t). See text. 0 Identified as PTH-Glu. h The carbohydrate is attached to this residue. i This peptide was obtained from a chymotryptic digest of HT30 (residues 345-355). 1 This peptide was obtained after one step of the Edman degradation of peptide HT30C1 (residues 345-349). k Isolated by diagonal electrophoresis (Tang and Hartley, 1967) of a tryptic and chymotryptic digest of Fc(t). * This peptide was obtained from a streptococcal proteinase-digest of HT32 (residues 361-370). n These peptides were isolated from a chymotrypsin C digest of HT33 (residues 371-392). n This peptide was obtained from a chymotryptic digest of HT33 (residues 371-392). The assignment of this residue is discussed in the text. (1
0
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COVALENT STRUCTURE OF TO-IMMUNOGLOBULIN.
Most of the assignments were straightforward, but eight peptides presented special difficulties which require discussion. The NHn-terminal tryptic peptide the of heavy chain, HT1 (residues 1-12, Figure 3A), contained one glutamic acid and two glutamines (Table 11). It had previously been established that residue 3 is glutamine (Cunningham er a[., 1970). Peptide HlClP2 (residues 5-10; Figure 3A, Table 11) contained one residue each of glutamine and glutamic acid. Treatment of this peptide with carboxypeptidase A at pH 5.5 released one residue of glutamic acid (residue lo), and digestion with leucine aminopeptidase released one residue each of valine, glutamine (residue 6), and serine. Peptide HT6a (residues 39-48, Figure 3B) contained one glutamic acid and two glutamines. PTH-Gln was identified after one step of the Edman degradation, indicating that residue 39 is glutamine. Digestion of HT6a with carboxypeptidase A at pH 8 released one residue each of homoserine and tryptophan. After lowering the pH to 5.5 and the addition of more enzyme, 0.7 residue of glutamic acid (position 46) was released after 6 hr. Therefore, residue 46 is glutamic acid and residue 43 must be glutamine. Peptide HT8a-LAP (residues 73-81, Figure 3C) contained one residue each of aspartic acid, glutamic acid, and asparagine, Residues 73 and 74 were assigned as aspartic acid and glutamic acid because PTH-Asp and PTH-Glu wereidentified after the first and second steps, respectively, of Edman degradation. Therefore, residue 77 was assigned as asparagine. Electrophoresis of a chymotryptic digest of HT9 gave peptide HT9Ca (residues 96-109, Figure 3D), which had a net charge of -2. Residues 106 and 107 were determined as PTH-Glu by Edman degradation of peptide HT9C3 (residues 104-121). Therefore residue 109 must be asparagine. Digestion of peptide HTl2Cl (residues 148-158, Figure 3E) with aminopeptidase M released the aspartic acid at position 148. Because the net charge of this peptide was -2, residue 152 must be glutamic acid. The remaining data in Table I1 complete the assignment of asparaginyl and glutaminyl residues in the region corresponding to CNBr fragments H I through H4. Similar data for the region corresponding to CNBr fragments H6through H7 are given in Table 111. The assignment of asparaginyl and glutaminyl residues in the region from residue 256 to 274 (peptide HT18, Figure 3F, Table 111) depended on the isolation of smaller fragments corresponding to this region. Peptides from a streptococcal proteinase digest of HT18 (Figure 3F, Table 111) were separated by ion-exchange chromatography on AG50X4. Peptide SP1 (residues 256-260) had a net charge of - 1, and therefore residue 258 must be glutamic acid. Pronase and aminopeptidase M digestion of peptide SP2 (Figure 3F, residues 262265) released 0.7 residue of aspartic acid (residue 265, Table 111). Peptide SP-3 (Figure 3F, residues 268-272) had a net charge of -1 at pH 4.7 indicating that two residues must be present as free acids. Treatment of this peptide with aminopeptidase M released 0.9 residue of glutamic acid in accord with the sequence His-Glu-Asx-Pro-Glx. The electrophoretic mobility of peptide HT18c (Figure 3F) indicated that residue 272 is glutamine; residue 270 therefore must be aspartic acid. Peptide HT18c was isolated in high yield from a tryptic digest of Fc(t). As tryptic cleavage of an Asp-Pro bond seems unlikely, this peptide probably resulted from nonenzymatic cleavage (E. L. Smith, 1969, personal communication) during
I X
the isolation procedures. Digestion of peptide H5C2 (residues 276-285, Figure 3G) with pronase and aminopeptidase M released 0.9 residue of asparagine and 0.9 residue of aspartic acid. For unknown reasons, neither glutamic acid nor glutamine was released, A similar digestion of peptide H5C2a (residues 276278, Figure 3G, Table 111) showed that residue 276 is asparagine, and therefore residue 280 must be aspartic acid. Peptide H5C2, which contained one histidine, was neutral at pH 4.7, indicating that residue 283 is glutamine. In order to determine the positions of the asparagine and glutamine in the region from residues 371 to 392 (Figure 3H), peptide HT33 was digested with chymotrypsin C. The resulting peptides (CC1 to CC5, Figure 3H) were fractionated by chromatography on AG5OX4. The assignments of aspartic acid to position 376, glutamic acid to positions 380 and 382, and asparagine to positions 384 and 390 were straightforward from the amino acid compositions (Table 111) of pronase and aminopeptidase M digests of peptides CCl, CC2, CC3, CC4, and CC5 (Figure 3H). Because peptide HT33C1 (Figure 3H) had a net charge of -3 at pH 6.5 (Table 111), the remaining positions (residues 385, 387, and 389) must be the free acids. To confirm these assignments, PTH-glutamic acid was identified from the Edman degradation of HT33C1 in the positions corresponding to residues 387 and 389. Digestion of peptide HT37 (Figure 31) with pronase and aminopeptidase M yielded two residues of asparagine, one residue of glutamic acid, and three residues of glutamine (Table 111). Residues 421 and 434 therefore are asparagine. The amino acid composition and net charge of peptide HT37bC1 (Figure 31, Table 111) indicate that residue 430 is glutamic acid; thus residues 418, 419, and 438 must be glutamine. The amino acid composition of peptide HT37bC3 (Table 111) confirms these assignments. Together with the data in Table 111, this completes the assignments of the asparaginyl and glutaminyl residues in the light and heavy chains of protein Eu. Some sources of possible error are considered below. Many of these studies were carried out on peptides which were obtained from CNBr fragments and thus had been exposed to 7 0 z formic acid for 4 hr at room temperature. In addition, many purification steps were required to obtain some of the peptides. Therefore, it is possible that some amide groups were lost prior to analysis. No evidence indicates that such deamidation occurred to any significant extent. The sequence Asx-Gly appears in three places in the Eu heavy chain (residues 109-110, 385-386, and 401-402). In the determination of the amino acid sequences of peptides containing residues 109, 385, and 401, the Edman degradation was severely limited or actually halted at these positions. This suggests that the rearrangement of the a-peptide bond to the /3 peptide or the corresponding imide (see Weber and Konigsberg, 1967; also Jackson and Hirs, 1970) occurs readily. Although this rearrangement probably took place during the Edman degradation, we cannot rigorously exclude the possibility that rearrangement occurred during the isolation of the peptides. Such a rearrangement may alter the electrophoretic mobility of a peptide and hence we assign residue 109 as asparagine and residues 385 and 401 as aspartic acid with some reservation. Of the 116 positions in the sequence of Eu at which the assignment of an amide or a free acid was required, 23 were
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made solely on the basis of electrophoretic mobility. Mobilities were measured for a total of 49 peptides. The observed mobility of each peptide was within ten per cent of the calculated mobility in all but three cases. For these three, additional evidence was obtained to confirm the assignments. In no case did the electrophoretic mobility of a peptide suggest an assignment different from that indicated by other methods.
W. H., and Edelman, G. M.(1968),Biochemistry 7,1973. Gerwin, B. I., Stein, W. H., and Moore, S. (1966), J . Biol. Chem. 241,3331. Gottlieb, P. D.,Cunningham, B. A., Rutishauser, U., and Edelman, G . M. (1970), Biochemistry 9,3155. Jackson, R. L.,and Hirs, C. H. W. (1970), J . Biol. Chem.
245,637. Jeppsson, J. O.,and Sjoquist, J. (1967), Anal. Biochem. 18,
264.
References Benson, J. V., Jr., Gordon, M. J., and Patterson, J. A.(1967), Anal. Biochem. 18,228. Cunningham, B. A,, Gottlieb, P. D., Konigsberg, W. H., and Edelman, G . M. (1968),Biochemistry 7,1983. Cunningham, B. A., Rutishauser, U., Gall, W. E., Gottlieb, P. D., Waxdal, M. J., and Edelman, G.M. (1970), Biochemistry 9,3161. Edelman, G. M., Gall, W. E., Waxdal, M. J., and Konigsberg, W. H. (1968), Biochemistry 7,1950. Gall, W. E., Cunningham, B. A., Waxdal, M. J., Konigsberg,
Offord,R. E. (1966),Nature (London)211,591. Rutishauser, U.,Cunningham, B. A., Bennett, C., Konigsberg, W. H., and Edelman, G.M. (1970), Biochemistry 9,
3171. Schwartz, J. H., and Edelman, G . M. (1963),J . Exp. Med.
118,41. Tang, J., and Hartley, B. S. (1967),Biochem. J . 102,593. Toda, H., and Folk, J. D. (1969), Biochim. Biophys. Acta
175,427. Weber, K., and Konigsberg, W. H. (1967), J . Biol. Chem.
242,3563.
The Covalent Structure of a Human yG-Immunoglobulin. X. Intrachain Disulfide Bonds* W. Einar Gall and Gerald M. Edelman
ABSTRACT: The arrangement of the 12 intrachain disulfide bonds in the human yG-immunoglobulin Eu has been determined. The first intrachain bond in each light ( K ) chain is formed by half-cystinyl residues at positions 23 (designated I, numbering from the NH2terminus) and 88 (11), and the second is formed by half-cystines at positions 134 (111) and 194 (IV). The four intrachain bonds in each heavy (71)chain are formed by half-cystines at positions 22 (Iand ) 96 (11), 144 (111) and
A
200 (IV), 261 (VIII) and 321 OX), and 367 (X)and 425 (XI). Each disulfide loop in the CL and CE regions of the molecule contains from 57 to 61 residues; the VL loop contains 66 residues and the VH loop contains 75 residues. These data indicate that the intrachain bonds form loops which have a linear and periodic arrangement and provide additional evidence for the hypothesis that immunoglobulins evolved by gene duplication.
previous paper in this series (Gall et al., 1968) described the location and arrangement of the interchain disulfide bonds in the human yGl-immunoglobulin Eu. A linear arrangement of all of the intrachain disulfide bonds of the molecule was first proposed by Waxdal et al. (1967). We now present proof of this arrangement. Peptides containing intrachain disulfide bonds have been isolated from Bence-Jones proteins and light chains of other immunoglobulins by Milstein and coworkers (for a review, see Milstein, 1966b), and the location of these peptides in the light chains has been suggested by comparison with amino
acid sequences obtained in several laboratories. Peptides containing some of the intrachain disulfide bonds have been isolated from the four subclasses of y chains (Frangione and Milstein, 1967; Pink and Milstein, 1967; Frangione et al., 1968,1969), and these authors also suggested a linear arrangement for these bonds. The results of the present studies, together with those on the interchain bonds (Gall et al., 1968) and the determination of the complete amino acid sequence of prodin Eu (Cunningham et al., 1968;Gottlieb et al., 1970; Cunningham et al., 1970; Rutishauser et al., 1970), establish the exact location of all of the disulfide bonds in the molecule.
* From The Rockefeller University, New York, New York 10021. Received December 8, 1969. This work was supported in part by grants from the National Science Foundation (GB 8371) and the U . S. Public Health Service (AM 04256 and AI 09273).
Materials and Methods
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Purification of the immunoglobulin Eu and preparation of the CNBr fragments have been described (Edelman et al.,
